蛋白激酶B在佛波酯诱导人胃癌细胞系凋亡中的作用
THE ROLE OF PROTEIN KINASE B IN THE APOPTOSIS OF GASTRIC CANCER CELLS INDUCED BY 12-O-TETRADECANOYLPHORBOL-1,3-ACETATE
Abstract
目的探讨佛波酯(TPA)诱导胃癌细胞系凋亡过程中蛋白激酶B(PKB)的作用。方法通过BrdU处理,流式细胞术检测以及DAPI染色,荧光显微镜观察分析TPA对胃癌BGC-823细胞的影响;免疫印迹法检测TPA对PKB蛋白表达水平、磷酸化的影响;核浆分离获得核浆蛋白,用于免疫印迹法检测TPA对胃癌细胞内PKB和其Ser 473位点磷酸化的核浆表达影响,以及激光扫描共焦显微镜观察TPA是否改变胃癌细胞内PKB的分布。结果TPA诱导BGC-823细胞凋亡;TPA下调BGC-823细胞内PKB蛋白表达,并且与TPA作用时间和TPA浓度呈正相关,与PP2A降解作用无关;TPA抑制PKB的Ser 473位点磷酸化,而对其Thr 308位点磷酸化没有明显影响;TPA下调细胞核内PKB的表达以及Ser 473位点的磷酸化;TPA并不改变PKB在胃癌细胞内的分布。结论PKB的抑制作用可能参与TPA诱导胃癌细胞凋亡过程;由TPA诱导的胃癌细胞凋亡可能部分是由于细胞核内PKB蛋白表达和Ser 473位点磷酸化下降所致。
【英文摘要】 Objective To study the role of protein kinase B(PKB) in gastric cancer BGC-823 cells treated by TPA. Methods Flow cytomety(FAC) and fluorescence microscope were used to detect the effect of TPA on gastric cancer BGC-823 cells,after cells were treated by BrdU and stained by DAPI,respectively.The effect of TPA on the expression level of PKB protein and its phosphorylation of Ser 473 and Thr 308 were investigated by Western blotting.When nuclear and cytoplasmic protein fractions were prepared through lysis of ...